Anticoagulants are used in the treatment of a wide variety of thrombotic disorders and in the laboratory to prevent the clotting of conserved blood. Heparin, for example, is widely used against thrombosis, but administration of heparin carries a high risk of hemorrhage or thrombocytopenia; is ineffective in many conditions, and may produce unpredictable results including recurrent thromboembolism. Derivatives of 4-hydroxycoumarin or of 1,4 idanedione also have a number of disadvantages. It is difficult to maintain a therapeutic dose due to a wide variability in the patient's diet, effective drug dose, and drug metabolism. Hemorrhage is a common complication and treatment of this side effect which may require the use of potentially hazardous (virus containing) plasma.
Other anticoagulants include those endogenous to the plasma, for example, antithrombin III, the extrinsic pathway inhibitor, and proteins obtained from certain plants and organisms, including the leech-derived polypeptides provided by U.S. Pat. No. 4,971,100 and Kunitz inhibitor obtained from soybean. The latter blocks the blood coagulation cascade by inhibition of activated Factor X, but the specificity of the inhibitor is so low that many side effects develop, including for example, inhibition of plasma kallikrein, plasmin and trypsin. Other active compounds, such as the Ascaris or Kazals inhibitor have also been criticized for their lack of specificity.
The complexity of the blood coagulation system exacerbates the need for an acceptable pharmaceutical agent capable of inhibiting hemostasis at a specifically defined point in the pathway. Clotting results from a complex series of interactions, which culminate in the thrombin-mediated cleavage of fibrinogen to fibrin and its subsequent crosslinking (1). Thrombin production may result from either of two pathways, an "intrinsic" pathway based on circulating blood components and an "extrinsic" pathway requiring a tissue-derived component. In each system, there is a cascade of reactions during which each of a series of inactive factors is converted by a proteolytic reaction into the corresponding active or "a" factor that is itself a proteolytically active enzyme or a non-proteolytically active cofactor capable of effecting the next step in the reaction cascade.
In the intrinsic activation pathway, circulating Factor XII (Hageman Factor) is postulated to bind to negatively charged surfaces or damaged aggregated platelets. Kallikrein cleaves Factor XII to form Factor XIIa. Factor XIIa, in the presence of high molecular weight kininogen, 1) cleaves circulating Factor XI (plasma thromboplastin antecedent) to Factor XIa (plasma thromboplastin) and 2) cleaves circulating prekallikrein to active kallikrein, effecting amplification of the pathway. Factor XIa, in the presence of calcium ions, cleaves circulating Factor IX (Christmas Factor) to Factor IXa Factor IXa, with circulating Factor VIII (antihemophilic globulin or AHG) in the presence of calcium ions and cell-derived phospholipid, forms a lipoprotein complex with circulating Factor X and cleaves it to form Factor Xa. Thus, in the intrinsic system, activation of Factor X requires the interaction of Factor IXa (enzyme) and Factor VIIIa (nonproteolytic cofactor) on a phospholipid surface in the presence of calcium ion.
In the extrinsic activation pathway, Factor VII associates with an integral tissue lipoprotein called tissue thromboplastin or tissue factor (Factor III) and in the presence of calcium ions, forms a complex with circulating Factor X and cleaves it to form a second source of Factor Xa (2). Factor Xa cleaves tissue factor-bound Factor VII to Factor VIIa, a much more active enzyme, thus amplifying the effects of this pathway. Factor IX may also be converted to Factor IXa by Factor VIIa and tissue factor further amplifying Factor X activation through the "intrinsic" activation pathway. Thus, the extrinsic Factor X activation complex is composed of Factor VII/VIIa (enzyme) assembled with the integral membrane-bound non-proteolytic cofactor, tissue factor, in the presence of calcium ions. The extrinsic pathway of activation is probably the physiologically relevant system, as patients deficient in Factor XII, prekallikrein or high molecular weight kininogen do not have bleeding disorders. Deficiencies in the other blood coagulation components may all lead to hemophilia.
Factor X is activated by either the intrinsic or extrinsic activation pathways. Factor Xa, with activated Factor V and in the presence of calcium ions and cellular phospholipid, forms a lipoprotein complex with Factor II (prothrombin) and cleaves it to form thrombin (Factor IIa). In other words, Factor Xa (enzyme) associates with Factor Va (nonproteolytic cofactor) in a macromolecular membrane complex responsible for the activation of prothrombin.
Thrombin converts circulating fibrinogen to the insoluble form fibrin, which spontaneously polymerizes into filaments and is then crosslinked under the action of Factor XIIIa, an enzyme formed from Factor XIII by thrombin activation.
There is a considerable body of evidence that tissue factor is the major initiator of coagulation. Following injury to a vessel, the contact of blood with tissue factor may be a primary event in the hemostatic and thrombotic processes. Indeed, recent studies suggest that tissue factor activation of the extrinsic pathway of coagulation is the primary early event mediating the formation of fibrin and the arrest of bleeding (3,4).
As indicated above, because of the complexity of the blood coagulation system, the preferred anticoagulant is one that can be definitively targeted to a precise and specific early stage in the blood coagulation cascade. However, as stated above, in the past, such agents were not available or suffered from serious drawbacks, such as their toxicity or antigenicity in humans.